The proband had unremarkable prenatal history, and a hearing test at birth was normal. In the first year of life, she had a history of recurrent ear infections and poor weight gain. She was referred to the pediatric nephrology clinic at the age of 12 months for evaluation of failure to thrive and anemia. The anemia was normocytic and normochromic. It was refractory to iron therapy. On physical examination, her growth parameters were as follows: weight 7.63 Kg at the 9th %ile, length 71.5 cm at the 16th %ile, head circumference 44.5 cm at the 38th %ile, and weight for length at the 14th %ile. Her vital signs were normal for her age, including her blood pressure (104/60 mmHg). The neurological examination revealed a hypotonic child with bilateral horizontal nystagmus, truncal ataxia, bilateral upper and lower extremity stiffness, and bilateral hyperreflexia (deep tendon reflexes at the biceps, brachioradialis, triceps, knee, and ankle were symptrical 4+ with clonus). The rest of the examination was unremarkable. The baseline investigations revealed normal electrolytes (Na 138, K 4.5, and Cl 105, all in mmol/L), normal Ca 2.5 and Mg 0.85, slightly elevated PO4 1.53 (all in mmol/L), normal serum albumin (39 g/L), and normal uric acid (0.59 mmol/L). The serum creatinine was slightly elevated (59 umol/L), with an estimated glomerular filtration rate (GFR) of around 55 mL/min/1.73 m² using the modified Schwartz formula. The urinalysis was unremarkable, but the specific gravity was consistent with hyposthenuria (SG 1.007, maximum recorded 1.014), which is consistent with urinary concentrating defects. The baseline KUB ultrasound revealed normal-sized kidneys with a small right cystic lesion (0.36 × 0.32 cm), with no other urogenital malformations but significance for bilateral medullary nephrocalcinosis. Further evaluation of the nephrocalcinosis revealed normal urinary calcium excretion (U Ca/Create ratio <0.2 mmol/mmol) and high urinary magnesium excretion (FEMg varied between 10% and 20% when hypomagnesemia). The rest of the metabolic stone workup was normal for her age (including urine oxalate, citrate, and uric acid). Subsequently, abdominal CT was performed that ruled out nephrocalcinosis but revealed bilateral multiple small-subcentimeter cystic lesions in the corticomedullary junction, suggestive of medullary cystic kidney disease. She was commenced on an erythropoietin-stimulating agent (ESA, darbepoetin) with a good response. She had regular follow-up sessions at the pediatric nephrology clinic every 12–16 weeks. She showed other manifestations of chronic kidney disease, including hyperphosphatemia, secondary hyperparathyroidism (peak PTH 14 pmol/L), and fluctuations of the kidney function that worsened with acute illnesses and dehydration. She was maintained on supportive therapy with ESA and vitamin D supplements, including calcitriol, to control the biochemical parameters of CKD.

At 2 years of age, the auditory brainstem response (ABR) showed severe sensorineural hearing loss, and the eye examination showed hypermetropia and horizontal gaze abnormality.

Regarding her developmental history, she walked after the age of 2 years, unsteadily, and then her balance gradually worsened; she lost her ability to walk at around 3 years of age, but she was still able to cruise around furniture and crawl. At 4 years of age, her condition was as follows: unsteady while sitting or standing but able to take few steps with the support of both hands, with her head flexed and back bent forward and wide-based, but subsequently, she became wheelchair-bound. Fine motor disability resulted in difficulties while trying to use her hands, and she needed help with all activities regarding her speech and language, as reported by her parents at 1 year of age; at 3 years of age, she only spoke single words, no sentences, but understood commands. Currently, she is able to sit without support but unable to stand, walk, cruise, use her hands, or speak clear words; she only speaks random words.

She had a history of recurrent gastroenteritis, upper respiratory infections, and ear infections. The recurrent infections were attributed to recurrent aspirations due to swallowing difficulties. She had undergone gastrostomy tube insertion at the age of 4 years to promote weight gain and minimize the risks of recurrent infections.

On physical examination at 9 years of age, her blood pressure was 98/70 (95th %ile for age and height: 112/74), height was 106 cm < the 3rd %ile for age, and weight was 13.6 Kg < the 3rd %ile. She showed no signs of acute distress. She was malnourished. Her pupils were equal and reactive to light, with limited horizontal eye movement. She tended to move her head instead of her eyes to look at things, which is suggestive of oculomotor apraxia, nystagmus, constant facial mimicking, dystonic arm movements, truncal hypotonia, increase in tone and brisk reflexes in the upper and lower limbs, and severe muscle wasting. Her skin showed a cutaneous hemangioma on the left side of the chest. The rest of the examination was unremarkable.

The most recent laboratory investigations revealed slightly impaired kidney function (serum creatinine 47 umol/L, with an estimated GFR 88 mL/min/1.73 m²), mild hyperparathyroidism (PTH 7.4 pmol/L), and normal serum calcium and phosphate levels. Her most recent hemoglobin level was normal (118 g/L).

Brain MRI at 2 years of age demonstrated prominent extra-axial spaces of both anterior temporal regions along with prominence of the subarachnoid spaces of the frontal, temporal, and parietal lobes. In addition, bilateral moderate cerebellar hemispheric and vermian atrophy were noted with ex vacuo dilatation of the fourth ventricle and enlargement of the retro-cerebellar fossa. In addition, the posterior part of the corpus callosum was oriented vertically. The FLAIR weighted images did not demonstrate any abnormal signal in the supratentorial and/or infratentorial regions (Figure 1) (Figure 1A, sagittal T1-weighted image, and Figure 1B, T2-weighted image) Figure 1B. Symptoms of a renal disease commenced around the time she was 17 months old, including polyuria and polydipsia, poor weight gain, and constipation. Ultrasound of both kidneys revealed increased parenchymal echogenicity with medullary calcinosis and reduced corticomedullary differentiation consistent with medullary cystic disease complex. Her neurological condition is progressing slowly. She has been followed up in the clinic on a regular basis for managing various comorbidities.

Patient 2 is the younger sibling of the proband. He had unremarkable perinatal history. He presented to the pediatric neurology clinic at 16 months of age with developmental delay, hearing loss, and failure to thrive. His head circumference was 47 cm (32 percentile for age), length was 72 cm, and weight was 8 kg below the 3 percentiles. He had a normal neurological examination apart from mild hypotonia. Based on the positive family history, a US scan of the abdomen performed at 17 months of age showed nephrocalcinosis. The patient was referred to the pediatric nephrology clinic at the age of 18 months for evaluation of bilateral medullary nephrocalcinosis. He had polyuria, polydipsia, and chronic constipation. No other associated symptoms were noted, including urinary symptoms. On physical examination, his weight was 7.6 Kg at <3rd %ile, length was 70 cm at <3rd %ile, head circumference was 47 cm at 76th %ile, and weight for length was at the 70th %ile. His vital signs were normal for his age, but he had high blood pressure for his age and length (110/70 mmHg, 95th %ile for age 102/54 mmHg). The neurological examination revealed a hypotonic child with bilateral upper and lower extremity stiffness and bilateral hyperreflexia (deep tendon reflexes at the biceps, brachioradialis, triceps, knee, and ankle were symptrical 4+ with clonus). The rest of the examination was unremarkable. The baseline investigations revealed normal electrolytes (Na 138, K 4.4, Cl 101, and HCO3 25, all in mmol/L), mild hypercalcemia (Ca 2.68 mmol/L), mild hyperphosphatemia (PO4 1.62 mmol/L), normal Mg (0.89), and normal serum albumin (44 g/L). The serum creatinine was normal (44 umol/L), but the estimated glomerular filtration rate (GFR) was 75 mL/min/1.73 m² using the modified Schwartz formula. He had mild normocytic and normochromic anemia (hemoglobin 104 g/L). The urinalysis was unremarkable, but the specific gravity was consistent with hyposthenuria (SG 1.016, varied between 1.010 and 1.016), which is consistent with urinary concentrating defects. The baseline KUB ultrasound revealed normal-size kidneys without other urogenital malformations but significant for bilateral medullary nephrocalcinosis. Given the strong family history of medullary cystic kidney disease in his elder sibling and being born to consanguineous parents, he was suspected to have the same kidney disease. Further evaluation of the nephrocalcinosis revealed normal urinary calcium excretion (U Ca/Create ratio <0.2 mmol/mmol). He was commenced on an erythropoietin-stimulating agent (ESA, darbepoetin) with a good response. He had regular follow-up sessions at the pediatric nephrology clinic every 12–16 weeks. He showed other manifestations of chronic kidney disease including hyperphosphatemia, secondary hyperparathyroidism (peak PTH 10 pmol/L), and fluctuations of the kidney function that worsened with acute illnesses and dehydration. He was maintained on supportive therapy with ESA and vitamin D supplements, including calcitriol, to control the biochemical parameters of CKD. He developed hypertension at the age of 6 years. He was hospitalized twice due to hypertensive emergency (BP up to 160/110 mmHg). He is maintained on dual antihypertensive medications (amlodipine and lisinopril) with good control.

Regarding developmental history, he started to sit at around 16 months of age and was able to walk at around 2 years of age, but his balance got worse, and eventually, he was unable to stand. Currently, he can sit without support, shuffling with his bottom, and can stand with support. His fine motor evaluation revealed the following: does coloring, can undress himself but cannot dress himself, can feed himself, can use a spoon, has delayed speech but can form sentences, receptive language is better, can recognize body parts, gender, and color, and has no behavioral problems. He had a history of recurrent infections, for which he was evaluated by a pediatric immunologist. He had several hospital admissions, ranging from 5–12 admissions per year, with different infections, such as recurrent pharyngitis, pneumonia, cellulitis, and bacterial meningitis. Immunological workup was normal, including immunoglobulins, immunoglobulin subclasses, lymphocyte subsets, neutrophil burst, CH50 at 62 U/mL (ref. range 30–75), and no neutropenia or lymphopenia. He was evaluated by a pediatric gastroenterologist for constipation, swallowing difficulties, and chronic/recurrent pancreatitis. The workup of pancreatitis was as follows: MRI MRCP was normal, abdominal US scan showed increased periportal echoes in the liver, no evidence of any focal lesion was seen, and no obvious intrahepatic biliary radicle dilation was detected. Lipase ranged from 52 to 221 IU/L (ref: 13–60), amylase ranged from 152 to 290 IU/L (ref: 28–100), liver enzymes were normal, and IgG 4 was more than 2.6 g/L (ref; 0.01–1.70). He also had an endoscopy, which showed multiple healed ulcers in the duodenum. A swallowing study showed that he had difficulties in swallowing thin liquids. On physical examination, his weight was 15.2 kg below the 3rd percentile, and he showed no signs of acute distress. He was wheelchair-bound, underweight, not dysmorphic, with equal pupils that were reactive to light, nystagmus, ptosis, and absent tracking. He tended to move his head instead of his eyes to look at things, which is suggestive of oculomotor apraxia. Respiratory examination revealed pectus excavatum and a small nodule on the lower rib anteriorly right to the sternum. His cardiovascular rate and peripheral perfusion were normal, and he did not have edema. His gastrointestinal pathway was soft, non-tender, and non-distended. Organomegaly was absent. He had unsteady mobility/gait and required assistance for balance. He had equinus deformity in both feet and contractures along the knees and elbows. He was neurologically alert but unable to move his eyes. He had mild stiffness in both elbows and knees, and bilateral limb contractures were noted. His DTRs were not brisk, and plantar reflex was bilaterally equivocal. Cognition and speech evaluation revealed that he could produce minimal speech, single words only.

Brain MRI at 18 months of age demonstrated mildly prominent extra-axial spaces of the frontal regions bilaterally associated with prominence of the subarachnoid spaces of the frontal, temporal, and parietal lobes and prominence of the ventricular system. In addition, thinning of the corpus callosum and cerebellar atrophy with enlarged retro-cerebellar fossa were present (Figure 1) (Figure 1C, sagittal T1-weighted image, and Figure 1D, T2-weighted image). Similar to patient 1, no abnormal signal was depicted on FLAIR weighted images. CT scan of the abdomen findings was consistent with medullary cystic disease complex (Figure 2). His condition has been progressing slowly, with a few preserved motor and verbal skills.

A comparison of the clinical phenotypes of the proband and her sibling with all the patients reported so far, including the Bedouin kindred (Perez et al., 2017), is provided in Table 1.

Chromosomal microarray analysis of the index patient and her brother revealed normal female and male patterns, respectively, and did not detect any copy number alterations relevant to the patient’s presentation. However, multiple regions of copy-neutral absence of heterozygosity (AOH) were detected in the patients, several of which were shared. The AOH segments harbored potential candidate genes associated with autosomal recessive conditions, such as nephronophthisis (NPHP1, WDR19, and TMEM67). All the AOH regions shared between the siblings are provided in Supplementary Table S1. The AOH region containing the nephronophthisis-related gene WDR19 on chromosome 4 was the biggest block of AOH (22.3 Mb) shared between the siblings.

Whole-exome sequencing was performed on the index patient, affected sibling, and parents using the TruSeq DNA Exome (Illumina Inc., San Diego, CA, United States) Library Preparation Kit and paired-end sequencing on the NovaSeq 6000 platform (Illumina Inc., San Diego, CA, United States). All the samples had a mean depth of coverage >125X, with 98% of the targeted regions covering at least 1X depth. Homozygous variants in genes associated with known autosomal recessive syndromes and shared between the affected siblings were prioritized, given parental consanguinity and no prior history of the disease in the family. A homozygous deletion of three nucleotides c.1049_1051delCAG (p.Ala350del) was identified in the SLC30A9 (NM_006345.4) gene in both the patient and her affected brother. Both parents were heterozygous for this change. This variant was located on a shared homozygous region on 4p15.1q12 spanning 22 Mb (35131357-57450848) (Supplementary Table S1). Though this region harbored other potential candidate genes associated with conditions overlapping with the patients’ phenotypes, no variants were identified in any of those genes. Sanger sequencing confirmed the presence of the variant in the corresponding allelic state in both the patients and their parents (Figure 3B). Of the four unaffected siblings of the patients, two were heterozygous carriers of the variant, while the other two siblings had the wild-type allele. The segregation of the variant is presented in Figure 3A.

This variant results in a deletion of an alanine residue at position 350 of the SLC30A9 gene. This in-frame deletion was previously reported in six individuals from a Bedouin kindred affected with Birk–Landau–Perez syndrome (Perez et al., 2017). This variant is not present in large population catalogs, such as the gnomAD database (Karczewski et al., 2020) and Middle Eastern-specific databases (Koshy et al., 2017). The deleted amino acid is highly conserved and is located at the predicted cation-efflux domain of the mature SLC30A9 protein. In vitro functional studies employing transient expression of the mutant or wild-type in human neuroblastoma cells demonstrated a significant decrease in cytosolic free Zn²⁺ levels in cells expressing the mutant when compared with cells expressing the wild-type variant (Perez et al., 2017). Functional complementation of the p.Ala350Del variant in CRISPR-Cas9 knockout (SLC30A9 ^−/−) cell lines failed to rescue the functional defect induced by genetic ablation of SLC30A9, indicating that the p.Ala350Del variant represents a loss-of-function allele (Deng et al., 2021). Aggregating all the available evidence (PM2, PM1, PS3, and PP1_strong) from the existing literature and employing the guidelines specified by the American College of Medical Genetics and Genomics (ACMG), we classified the variant as pathogenic (Richards et al., 2015).